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Diss Factsheets

Administrative data

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25-11-2013 to 11-3-2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Objective of study:
metabolism
Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The objective of this study was to compare the metabolism in vitro of the test substance using cryopreserved hepatocytes from rat, rabbit and human. Incubations were conducted with cryopreserved hepatocytes in duplicate for each species at three test substance concentrations (5, 25 and 100 µM) over three incubation times (0, 1 and 4 h). A radio-HPLC method was developed and used to determine the metabolite profiles generated during the hepatocyte incubations with test substance.
GLP compliance:
yes (incl. QA statement)
Remarks:
Huntingdon Life Sciences, Huntingdon Research Centre, Woolley Road Alconbury, Huntingdon Cambridgeshire, PE28 4HS UK

Test material

Test material form:
liquid
Radiolabelling:
yes

Test animals

Details on test animals or test system and environmental conditions:
Rat (Sprague-Dawley), rabbit (New Zealand White) and human cryopreserved hepatocytes were obtained from Bioreclamation IVT (formerly Celsis IVT) and delivered stored frozen in liquid nitrogen. All hepatocytes were from male donors.

Administration / exposure

Route of administration:
other: in vitro application in liquid medium
Vehicle:
ethanol
Details on exposure:
- Species: Rat, rabbit and human
- Test substance concentrations: 5, 25 and 100 μM
- Incubation times: 0, 1 and 4 h
- Number of replicates: 2
- Cell concentration: 1 x 10^6 viable cells per mL of culture medium
- Volume of incubation medium: 1 mL
Duration and frequency of treatment / exposure:
0, 1 and 4 h
Details on study design:
INCUBATION CONDITIONS
- The incubation components were mixed together in glass vials so that each sample contained the following: Foetal calf serum (100 µL), Supplemented Williams’ Medium E (890 µL – volume of hepatocyte suspension), Hepatocyte suspension (volume containing 1 x 10^6 viable cells), Test substance (10 µL of solution in ethanol).
- The vials for the 0 h incubations were sealed immediately after preparation and the reactions stopped.
- At the end of the requisite incubation period, the reactions were stopped by transferring the sealed incubation vials on to solid carbon dioxide. The contents of the vials were allowed to freeze completely (minimum of approximately 10 min) prior to transfer to storage at approximately -20°C pending analysis.
-In addition, viability samples were incubated to assess LDH activity and leakage. These consisted of duplicate 0.5 mL incubations with hepatocytes (no test substance) from each species for 0, 1 and 4 h, together with single 0.5 mL incubations with hepatocytes from each species andtest substance (100 µM) for 1 and 4 h.
- the hepatocyte incubations with test substance were conducted in duplicate for each species (rat, rabbit and human), at each incubation time (0, 1 and 4 h) and at each concentration (5, 25 and 100 µM).

CONTROL INCUBATIONS
- Incubation of test substance for 0, 1 and 4 h in the absence of hepatocytes (singly at each concentration).
- Incubation with hepatocytes for 4 h in the absence of test substance (single sample per species). These samples were not analysed, but were prepared to permit a subsequent assessment of endogenous materials if required.
- Positive control samples incubating [14C]7-EC at a concentration of 50 µM for 4 h with and in the absence of hepatocytes (each conducted in duplicate).

MEASUREMENT OF LDH LEAKAGE
- Duplicate 0.5 mL incubations with hepatocytes (no test substance) from each species for 0, 1 and 4 h, together with single 0.5 mL incubations with hepatocytes from each species and the test substance (100 μM) for 1 and 4 h. The methodology for further treatment was adapted from Howes 1990.

ANALYSIS OF 7-ETHOXY[14C]COUMARIN INCUBATIONS
-Following storage at approximately -20°C, the 7-EC samples were centrifuged (18,620 x g, 15 min, 4°C). The resulting supernatants were transferred to clean tubes and evaporated to dryness using a centrifugal evaporator.
-The samples were reconstituted in 40 mM ammonium formate pH 5.0 (1 mL, with one exception where 1.35 mL was used was used to accommodate a heat control) and an aliquot (0.45 mL) of each was taken into a clean tube for use in deconjugation. The remainder of each reconstituted supernatant was transferred to a HPLC vial.
-Deconjugation was conducted by incubating (1 h, 37°C) the aliquots of the reconstituted supernatant with ß-glucuronidase enzyme (2000 units, type H1 from Helix pomatia, also containing sulfatase activity). Samples were then transferred to HPLC vials.
Positive controls for ß-glucuronidase and sulfatase enzyme activities were determined by the production of free phenolphthalein from phenolphthalein glucuronic acid and p-nitrocatechol from p-nitrocatechol sulfate, respectively, upon the addition of 1 M sodium hydroxide, after incubation (1 h, 37°C).
-The percentage of sample radioactivity associated with 7-EC and the known metabolite 7-hydroxycoumarin (7-HC) was determined for untreated and deconjugated samples by HPLC.
Details on dosing and sampling:
SAMPLE PREPARATION
Hepatocyte incubation samples (0, 1 and 4 h) were treated as follows (samples incubated in the absence of hepatocytes were analysed directly without filtration):
-Each sample was allowed to thaw on ice and then was vortex mixed briefly. Duplicate aliquots (50 µL for 5 and 25 µM incubations and 25 µL for 100 µM incubations) were taken into separate scintillation vials. Ultima Gold scintillation cocktail (approximately 5 mL) was added to each vial and the vials were submitted to LSC. An aliquot (approximately 250 µL) was transferred into a syringe body and was then passed through a Pall Acrodisc GHP filter (13 mm, 0.2 µm) directly into an HPLC vial.
-Duplicate aliquots (as above) of the filtrate were taken into separate scintillation vials, Ultima Gold scintillation cocktail (approximately 5 mL) was added to each vial and the vials were submitted to LSC to assess the recovery of radioactivity following filtration.
-Due to a relatively low recovery of radioactivity following filtration of the 0 h samples, the original incubation samples were allowed to thaw on ice and were then vortex mixed briefly. An aliquot (approximately 250 µL) of each was transferred into a microcentrifuge tube and centrifuged (15 min, 10,000 x g). Each supernatant was transferred into a HPLC vial and duplicate aliquots (50 µL for 5 and 25 µM incubations and 25 µL for 100 µM incubations) were taken into separate scintillation vials. Ultima Gold scintillation cocktail (approximately 5 mL) was added to each vial and the vials were submitted to LSC to assess the recovery of radioactivity following centrifugation.
-Samples were stored at approximately -20°C prior to and on completion of analysis and at approximately 10°C during analysis.

INSTRUMENTATION AND ANALYTICAL METHOD
- HPLC system: Waters Alliance 2695XE (including solvent conditioning module, pump, autosampler, column heater)
- Detectors: Waters 2487 UV detector LabLogic ß-RAM radioactivity detector
- Column: Phenomenex Kinetex C18, 2.6 µm, 100 Å, 100 × 4.6 mm
- Guard column: Phenomenex KrudKatcher + SecurityGuard Ultra C18
- Column oven temperature: 50°C
- Mobile phase A: 0.1% (v/v) formic acid in purified water
- Mobile phase B: 0.1% (v/v) formic acid in acetonitrile
- Flow rate: 1.5 mL/min
- Detection: UV at 220 nm and Radioactivity flow detector (with a 500 µL homogenous cell using Monoflow 4 liquid scintillant at 5 mL/min)
- Gradient: see any 'other information on materials and methods'
- Data capture time: 40 min
- Radioactivity measurements were performed by taking aliquots of samples (in duplicate where possible) by volume and mixed directly with Ultima Gold scintillation cocktail (5 mL) for analysis using Wallac 1409 automatic liquid scintillation counters.

Results and discussion

Metabolite characterisation studies

Metabolites identified:
no
Details on metabolites:
- The hepatocytes used in the study were shown to be metabolically viable over the incubation periods used.
- The metabolite profiles obtained following incubations of test substance with rat, rabbit and human hepatocytes and in the absence of hepatocytes showed up to 40 regions of radioactivity; assigned E1 to E40 in order of increasing retention time. Not all regions were observed in each radioactivity profile.
- The extent of metabolism following incubations of test substance with rat, rabbit and human hepatocytes generally increased with time and was therefore greatest for the 4 h incubations. See 'any other information on results' for an overview in tabular format.
- Similarities in the radioactivity profiles were observed across species and for the different incubation concentrations used. The profiles were relatively complex and showed a total of up to 40 regions of radioactivity, although not all were observed in all species and at each concentration. The number of regions tended to increase with incubation time. No regions of radioactivity were observed in the human profiles that were not also observed in profiles from at least one other species.
- Eight major metabolites (ie >10% of sample radioactivity) were detected most frequently in the profiles: Regions E17, E19, E20, E21, E30, E32, E34 and E35. Between two and five of these major metabolites were observed in most profiles.
- Overall, metabolism of the test substance was rapid, with between two and five major components typically observed following incubation with rat, rabbit and human hepatocytes. None of the metabolites observed were unique to the human hepatocyte profiles.

Any other information on results incl. tables

Summary of the radioactivity profiles obtained following 4h incubations

Region

RT (min)

 

Rat

Rabbit

Human

No hepatocytes

 

 

5µM

25µM

100µM

5µM

25µM

100µM

5 µM

25 µM

100 µM

5 µM

25 µM

100 µM

E1

0.8

-

-

-

o

o

o

o

-

-

o

-

-

E2

1.1

-

-

-

o

o

o

-

-

-

-

-

-

E3

2.4

-

-

-

-

-

-

-

-

-

-

-

-

E4

3.3

-

-

-

o

o

-

-

-

-

-

-

-

E5

3.8

o

-

o

-

-

-

-

-

-

-

-

-

E6

4.4

-

o

o

-

-

-

-

-

o

-

-

-

E7

5.0

-

-

-

-

-

-

-

-

-

-

-

-

E8

5.6

-

-

-

-

-

-

-

-

-

-

-

-

E9

6.1

o

o

o

o

o

o

-

-

-

-

-

-

E10

6.5

o

o

o

o

o

-

o

-

-

-

-

-

E11

7.5

o

-

o

o

o

o

o

-

-

-

-

-

E12

8.4

o

-

-

-

o

-

-

o

-

-

-

-

E13

9.0

o

-

-

o

-

-

-

-

-

-

-

-

E14

10.3

o

o

o

o

o

o

o

o

o

-

-

-

E15

11.3

o

o

-

o

-

-

o

o

-

-

-

-

E16

11.7

o

o

o

o

o

o

o

o

o

-

-

-

E17

12.6

♦

♦

♦

o

o

o

♦

♦

♦

-

-

-

E18

13.4

o

o

o

o

o

o

-

o

o

-

-

-

E19

13.7

o

o

o

♦

♦

♦

o

-

o

-

-

-

E20

15.0

♦

♦

♦

o

o

o

o

o

o

-

-

-

E21

16.2

o

o

o

♦

♦

♦

o

o

o

-

-

-

E22

17.4

-

-

-

-

-

-

-

o

o

-

-

-

E23

18.4

-

-

-

o

-

-

-

-

-

-

-

-

E24

19.0

-

-

-

-

-

-

-

-

-

-

-

-

E25

21.5

-

-

-

o

o

-

o

o

-

-

-

-

E26

22.3

-

-

-

-

o

-

-

-

-

-

-

-

E27

23.0

o

o

o

o

o

o

o

o

o

o

-

-

E28

24.2

o

♦

♦

o

o

o

o

o

o

-

-

-

E29

25.1

-

-

-

-

-

o

-

-

-

-

-

-

E30

25.6

o

o

♦

o

o

o

♦

♦

♦

-

-

-

E31

26.0

-

-

-

o

o

o

-

-

o

-

-

-

E32

26.6

-

-

-

o

o

o

-

-

o

-

-

-

E33

27.4

-

-

-

-

-

-

-

-

-

o

-

-

E34

27.8

♦

♦

♦

♦

♦

♦

♦

♦

♦

o

o

o

E35

28.6

-

-

-

o

o

o

-

-

o

-

o

o

E36

29.6

-

-

o

o

o

o

o

-

o

-

-

-

E37

30.4

-

-

-

-

-

-

o

-

-

♦

♦

♦

E38

31.6

-

-

-

o

o

-

o

o

-

♦

♦

♦

E39

32.6

-

-

-

-

-

-

-

-

-

-

-

-

E40

36.5

-

-

-

-

-

-

-

-

-

o

o

o

- Not detected or <0.5% of sample radioactivity; o  Minor (0.5% to 10% of sample radioactivity); ♦  Major (10% or more of sample radioactivity)

Applicant's summary and conclusion

Executive summary:

In vitro metabolism of the test substance using cryopreserved hepatocytes from rat, rabbit and human was assessed in a GLP study. Incubations were conducted with cryopreserved hepatocytes in duplicate for each species at three test substance concentrations (5, 25 and 100 µM) over three incubation times (0, 1 and 4 h). A radio-HPLC method was used to determine the metabolite profiles generated during the hepatocyte incubations with test substance. The hepatocytes used in the study were shown to be metabolically viable over the incubation periods used. Overall, metabolism of the test substance was rapid, with between two and five major components typically observed following incubation with rat, rabbit and human hepatocytes. Similarities in the radioactivity profiles were observed across species and for the different incubation concentrations used. The profiles were relatively complex and showed a total of up 40 regions of radioactivity, although not all were observed in all species and at each concentration. The number of regions tended to increase with incubation time. None of the metabolites observed were unique to the human hepatocyte profiles.